1. Field of Invention
Small-size essentially impermeable liposomes containing lecithin (a), which are dimensionally stable and non-aggregating, and the size of which is controlled and determined by the fatty acid-esterified lipoamino acid or lipopeptide (b) content thereof, their production, and their use, the liposome membranes consisting essentially of (a) and (b).
2. Prior Art
As is generally known, liposomes are small vesicles having a bilayer structure. They are used in the pharmaceutical area as drug vehicles, e.g., for parenteral or topical application.
The most varied agents with different chemico-physical properties can be incorporated into liposomes. Water-soluble agents can be located in the core and between the vesicle bilayers. Lipophilic substances, on the contrary, are incorporated in the bilayer. Therefore, the incorporation of substantial amounts of such agents into liposomes can result in a marked increase in vesicular diameter. In such case, pharmacokinetics and therefore also therapeutic properties can change considerably. Small liposomes afford more advantages because they are not identified by the RES (reticulo-endothelial system). In consequence, they are not eliminated from circulation through hepatic or renal filtration, resulting in higher plasma levels over prolonged periods of time. For example, for such small liposomes half-lives of over 20 hours were determined (cf. J. Senior et al., Biochem. Biophys. Acta 839, 1 (1985)). This means greater availability of an encapsulated active agent at the potential site of action, e.g., a tumor, because the circulating liposome deposit is very slowly degraded by enzymatic decomposition and interaction with other blood constituents. During this process the encapsulated active substance is released and is available for therapeutic utilization.
Large vesicles, on the contrary, are identified by the RES (hepatic Kupffer cells) as foreign bodies and destroyed. Even a few minutes after the i.v. application of such particles, 90% of the particles are eliminated. With the application of 400 nm liposomes, a half-life of about 10 min. was found; with particles of 1000 nm it was reduced to only about 5 min (cf. J. Senior et al. and P. O. Lelkes, Liposome Technology, Vol. 1). Also after topical application, small vesicles containing active ingredients may exert a better effect through improved penetration, for example, along the hair follicles. Additionally, an interaction may be possible with the natural bilayer structures in the intercellular gaps (cf. W. Curatolo, Pharm. Res. 4, 271-341 (1987), G. Grubauer et al., J. Lipid Res. 30, 89-96 (1989)). Furthermore, from a statistical point of view, the interaction of small unilamellar vesicles (SUV) with other cells is likely to be greater than that of multilamellar vesicles (MLV), which facilitates the transfer of membrane constituents, e.g., bilayer-bound ingredients.
In FR-A 2 591 105, pharmaceutical compositions based on lipid-containing multilamellar vesicles for dermatological or cosmetic application are described which contain a retinoid or an analogue thereof. This combination is supposed to prevent skin irritations. The vesicular constituents used are hydrated lipids and, for example, cholesterol and sitosterol. However, the vesicles of the resulting liposomes are large in particle size, and are accompanied by numerous expected disadvantages. Therefore, it is more reasonable to strive for smaller size liposomes.
In addition to particle size, the dimensional stability of the liposome preparations and the extent of their permeability are also decisively important for their effective therapeutic or cosmetic use.
The liposome membrane consists of conically-shaped lecithin and in the present case also fatty acid esterified collagen hydrolysate (FAECH) molecules. A bilayer association of these molecules always forms a curved plane, leading finally to the formation of hollow spheres. Since the intermolecular distances are irrefutable given by the physico-chemical nature of these compounds, the diameter of a liposome is directly related to the number of membrane-forming molecules.
An increase in vesicle size always implies an increase of the number of membrane-forming molecules per vesicle (because a liposome cannot swell). Since any liposome preparation is a closed system, such a dimensional increase as mentioned before can occur only by fusion of liposomes. This process results in the formation of a common membrane with twice the number of molecules and a greater diameter than each vesicle had before. Fusion of vesicles always results in a decrease in the number of liposomes in a preparation.
The benefit of liposomes in comparison with conventional topical formulations (such as creams, gels, ointments) is an enhanced penetration of the drug into deeper layers of the skin. The extent of penetration is dependent on size and number of the vesicles, the deepness of penetration on the size of the liposomes. The therapeutic effectiveness of a liposomal preparation increases with the number and the smallness of its vesicles.
During the fusion-process an at least partial release of an encapsulated (water-soluble) active ingredient, e.g., drug, takes place. Finally, fusion to some few large vesicles can also result in sedimentation of the liposomes in the preparation, thus leading to an inhomogeneous distribution of the active substance and, as a consequence, a reduced dose or an overdose of the encapsulated active ingredient.
To summarize, an increase in size is the first and main indicator of physical instability of liposomes. All the subsequent observations as sedimentation, permeability giving rise to a decrease in encapsulated drug, and inhomogeneous distribution and diminished therapeutic effectiveness of the liposomal preparation, are logical and inevitable consequences.
Exactly all these problems are solved by stabilizing lecithin liposomes according to the present invention with certain collagen hydrolysates, thus preventing the previously-inevitable gradual increase of vesicle size during storage.
As already described, liposomes mediate the penetration of a pharmaceutical agent into the skin or systemically. The degree of penetration and the amount of active substance mediated depends on the size of the liposomes. As compared to large liposomes, small liposomes are able to reach deeper skin layers and to transport a greater amount of substance into these layers or into a systemic target. Liposomes of a size smaller than 30 nm are even able to penetrate the skin, thereby entering circulation. In this case, of course, the liposome-mediated active substance does not act in the skin but has a systemic effect. On the contrary, large liposomes having a size of over 60 nm get caught in the upper skin layers where they release the active substance. From the therapeutic point of view, it is very important to be able to provide tailor-made vesicle sizes for specific indications.
For example, when the acid mantle of the skin is pathologically changed and the skin develops a tendency to drying out, scaling, reddening, etc. (neurodermitis), formulations containing relatively large liposomes (&gt;60 nm) should be employed to form an artificial protective lipid film on the skin surface.
In acne formulations, the liposomes should have a size between 35 and 50 nm. These vesicles penetrate deep into the skin and transport the pharmaceutical agent to its deeply located site of action, the pilosebaceous glands.
If a systemic effect is envisaged by using a liposomal formulation (insulin in diabetes mellitus; beta-blockers in the case of asthma/cardiac insufficiency), then the vesicles must penetrate the skin. For this purpose a liposomal size smaller than 30 nm is recommendable.
The production of such tailor-made liposomes by the present approach and method has so far not been described or even suggested. Vesicles of varying size could so far only be produced by means of different manufacturing technologies or by qualitatively changing the formulation (which means substitution or addition of individual ingredients).
In the present process, only one technology is required (high-pressure homogenization or ethanol injection) and the qualitative formulation remains unchanged; only the lecithin/lipoamino acid/lipopeptide proportions need be varied.
In all the aforementioned applications, the selected size of the liposomes must be stable. If there was a growth in particle size, the liposomes designed for systemic treatment would no longer be able to penetrate the skin, or liposomal acne formulations would remain on the skin. In both cases, the formulations would thus become ineffective.
Besides the possibility of, for the first time, producing tailor-made liposomes having a targeted size, especially designed to meet defined therapeutic requirements, the present invention, requiring combination with lecithin of a lipoamino acid or lipopeptide ester in the given concentration range, without more, ensures a stable size of the liposomes over the entire storage period.
From a galenical point of view, the therapeutic application of liposome preparations has often been problematic and limited because of their permeability, dimensional instability, and the particular active ingredient incorporated. One of the main problems is also a degradation of the active ingredient. Route and rate of decomposition are specific and vary from substance to substance. Oxidation processes can be reduced by using suitable antioxidants such as butyl-hydroxytoluene (BHT) or Vitamin E, whereas pH-dependent hydrolysis can be reduced by suitable buffers. In the case of substances sensitive to such procedure, hydrolysis can still be increased by intensified contact with water, but they can be stabilized by sufficient incorporation in the bilayer. Decisive for the stability of the active ingredient are the decomposition reaction involved and the physico-chemical properties of the substance.
Besides decomposition of the active ingredient, as described above, the liposomes themselves can be affected by lipid oxidation, lipid hydrolysis, and especially aggregation. Lipid oxidation can be reduced through addition of antioxidants as described by A. A. Hunt, S. Tsang, in Int. J. Pharm. 8, 101 (1981) or by A. W. T. Konings in Liposome Technology, Vol. I. Alternatively, there is the possibility of using lecithins with non-oxidizable saturated fatty acid ester groups which cannot be oxidized (cf. P. Kibat, Dissertation, University of Heidelberg, 1987). Hydrolysis of lecithins to lysolecithins can be reduced by buffering the system within a pH range between 6 and 7 (cf. S. Froeckjear et al., Optimization of Drug Delivery, A. Benzon Symposium 17, Kopenhagen (1982)).
Moreover, especially with small unilamellar vesicles (SUV), undesirable particle aggregation can occur already after a few weeks. During this process, the properties of the vesicles change and diminish the shelf-life of a product. The measures attempted so far to combat such changes have comprised the use of hydrated (saturated) lipids or cholesterol up to 50 mol-% in relation to the total lipid content (cf. P. Kibat). Such measures alone have not been successful. The technical processing of hydrated lipids is often more difficult, and they are accordingly less preferred than natural lecithins from eggs, soy beans, and the like.
When using cholesterol for stabilization purposes, the reduced bilayer-binding of lipophilic constituents is often problematic because of a competitive displacement through cholesterol (cf. Mentrup, Dissertation, University of Heidelberg, 1988), resulting in aggregation, particle dimension instability, and undesirable permeability and leakage, just as shown by Handjani (discussed hereinafter). Depending on the characteristics of the active ingredients, the bilayer-binding of therapeutically-effective amounts may be impossible in such a system.
Proceeding directly along such unacceptable lines, just as FR-A 2 591 105, is Handjani et al. published FRG application DE 3713493A1, published on Oct. 29, 1987, which employs a component A (a synthetic lipid) or possibly lecithin and a fatty acid esterified collagen hydrolysate, but also substantial amounts of cholesterol, in the production of vesicles or liposomes which, clearly according to Handjani, cannot contain higher than 15% of a fatty acid-esterified collagen hydrolysate since the impermeability of the vesicles should remain within a "tolerable range" and, "if the percentage is higher than 15%, the permeability of the vesicles is too pronounced leading to their dysfunction". This is moreover clear from the Table of Handjani, wherein vesicles were produced having a percentage of 20% by weight of a fatty acid ester of a collagen hydrolysate plus cholesterol, in which case the permeability rose from 13% at zero days to 24% at the end of fifteen days and from 31% at zero days to 57% at the end of fifteen days. Moreover, even with lesser amounts of the fatty acid esterified collagen hydrolysate and cholesterol, the permeability attained at the end of fifteen days was in almost every case totally unacceptable from the standpoint of storage stability and long-term effectiveness. Copies of the Handjani reference in German and a certified translation thereof into English have been furnished the Examiner during prosecution of the parent application. Handjani teaches that with increasing collagen hydrolysate content of the formulation, the permeability of the membrane reaches a level at which the stability of the dispersion is affected, the resulting fusion of the liposomes leading to increasing particle diameters.
Therefore, Handjani uses the collagen hydrolysate in a range between 1 and maximally 15%. Within this range, the topical properties are slightly improved as compared to the formulation not containing collagen hydrolysate but the liposomes are permeable to the encapsulated substance (see permeability values of 5-57% in the Table on page 7). A vesicle permeability of even 5% is totally unacceptable, especially when it commences at zero days.
Although this patent disclosure is mainly concerned with allegedly novel so-called "non-ionic amphiphilics", more properly designated "amphiphatics", namely, Handjani's synthetic lipid component A having an ether and polyglycerol structure, and although the vesicles or liposomes produced according to that patent disclosure are allegedly between 0.025 and 5 .mu.m, the fact is that the only liposomes or vesicles produced according to the Examples have a size of 0.5 .mu.m, 1 .mu.m, 1.0 .mu.m, 0.5 .mu.m, less than 1 .mu.m, 0.2 .mu.m, 1 .mu.m, 0.3 .mu.m, 1 .mu.m, 0.2 .mu.m, 0.2 .mu.m, and 0.2 .mu.m, which can hardly be considered "small" vesicles since 0.2 .mu.m is equivalent to 200 nm. The only example of this patent which employs lecithin is Example 3, which also employs one-third as much cholesterol as lecithin and only one-eighth as much of a fatty acid ester of a collagen hydrolysate, and produces a vesicle having a mean vesicle size "smaller than 1 .mu.m". In addition to producing only relatively large vesicles, this patent makes no disclosure or suggestion of any way in which vesicle size even might possibly be controlled or particle size reduced, much less by using increased ratios of fatty acid ester of a collagen hydrolysate, and still much less at any percentage greater than 15% of the lipid phase, at which percentage the patent states categorically that its vesicles become dysfunctional due to permeability. Example 3 of the Handjani reference, not unimportantly, shows the employment of the fatty acid-esterified collagen hydrolysate at 8%, the employment of cholesterol at 22%, and the employment of soy lecithin at 66%, said percentages being of the lipid phase and, as previously stated, only one-eighth as much fatty acid-esterified collagen hydrolysate as lecithin or approximately 12.5% thereof, whereas the cholesterol comprised one-third or 331/3% of the lecithin and almost three times the amount of the fatty acid esterified collagen hydrolysate.
Applicants have found that, when employing lecithin and a fatty acid esterified collagen hydrolysate of a particular type and a mixture consisting essentially thereof, and not cholesterol or a synthetic lipid of Handjani's type A, and providing a vesicle or liposome wall consisting essentially of the lecithin and the fatty acid ester of a collagen hydrolysate (FAECH) of the particular hereinafter-defined type, there is no problem in maintaining the impermeability of the vesicles produced and, moreover, the stability of the particles is at a maximum and aggregation is essentially non-existent so that optimum particle size, stability, non-aggregation, and impermeability are maintained in the vesicles or liposomes provided according to the present invention, quite to the contrary of any teaching of Handjani. Moreover, applicants have found that vesicle dimensional control and particle size reduction can be attained by employing relatively high ratios of the selected FAECH to lecithin, far beyond those which have been stated by Handjani to be inoperative and, in fact, amounts of FAECH to lecithin extending all the way from 10% to 90% of the lipid phase, in the absence of cholesterol or other of Handjani's ingredients which interfere with the desired results of the present applicants, are not only productive of small-size and entirely satisfactory vesicles from every conceivable standpoint but that ratios of the selected FAECH, of a particular type as hereinafter defined, to the lecithin (which are totally contraindicated and in fact stated to be inoperative by Handjani) can be employed to attain a particle size of the vesicles or liposomes produced in inverse proportion or relation to increased ratios of FAECH to lecithin, an important dimensional control which has not been taught, suggested, or even vaguely adumbrated by the prior art, but rather directly contraindicated thereby.